Fluorescent proteins that emit light in the presence of stimulating radiation in the absence of substrate have been used as research tools for many years. The best known and initially used such protein is the green fluorescent protein (GFP) isolated from a Aequorea victoria, but a large number of such proteins have been isolated from other sources or obtained synthetically which display a wide variety of emission maxima so that the historical term GFP has been used to describe proteins that appear in a full spectrum of visible color, including red, blue, and yellow. See, e.g., Delagrave, S., et al., BioTechnology (1995) 13:151-154; Heim, R., et al., Proc. Natl. Acad. Sci. USA (1994) 91:12501-12504. A number of organisms have been successfully modified to express such fluorescent proteins. These include Caenorhabditis elegans (Chalfie, M., et al., Science (1994) 263:802-805), Drosophila melanogaster (Wang, S., et al., Nature (1994) 369:400-403), zebrafish (Peters, K. G., et al., Dev. Biol. (1995) 171:252-257; Amsterdam, A., et al., Dev. Biol. (1995) 171:123-129), Dictyostelium and Arabidopsis thaliana (Sheen, J., et al., Plant J. (1995) 8:777-784; Hu, W., FEBS Lett. (1995) 369:331-334).
Okabe, et al., FEBS Lett. (1997) 407:313-319, have inserted the wild-type GFP into pCAGGS (containing the chicken beta-actin promoter and cytomegalovirus enhancer, beta-actin intron and bovine globin polyadenylation signal—Niwa, H., Gene (1991) 108:193-199) and produced transgenic mouse lines (Ikawa, M., FEBS Lett. (1995) 375:125-128; and Ikawa, M., Dev. Growth Diff. (1995) 37:455-459). Although a bright green light emission was observed in the muscle and pancreas of more than 20 of these transgenic mouse lines, GFP expression was not ubiquitous and light emission was not visible to the naked eye in other tissues. However, when a modified form (EGFP) was used in this expression system, the transgenic mice express the EGFP transgene in the entire body, from pre-implantation embryo to adult stages.
Wild-type eggs fertilized with green male sperm were not green at the 2-cell stage but subsequently became green after subsequent stages of embryogenesis. Newborns were green fluorescent. The blood vessels were classified as ‘bright’ in the EGFP-bearing lines. The hair of these animals was not green. Transgenic mice were uniformly green with the exception of hair and red blood cells. The brain, liver, kidney, adrenal gland and testis, lung, muscle, heart, intestine, and adipose tissue, thymus, spleen and testicular cells fluoresced green when irradiated with blue excitation light.
The transgenic mouse lines were normal despite a significant amount of EGFP expression; EGFP therefore is non-toxic.
One embodiment of the immunocompromised rodents exhibiting fluorescence described in the invention is reported in Yang, M., et al., Proc. Natl. Acad. Sci. USA (2003) 100:14259-14262 (November 25th issue).
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